This invention relates to an apparatus and a method of adjusting the focal point of an operating laser beam onto a selectable microscopic target region within an object, preferably a biological object, to be treated and observed under a microscope.
The treatment or destruction of material within a defined microscopically small region by a laser beam has become an important tool in scientific biological research. An example of its employment is the defined destruction of particular areas of cells in living tissues such as cell nuclei, chromosomes, etc., for the purposes of investigating the result of such cellular damage or for analyzing the composition of the materials that are vaporized by such treatment. In this and similar applications, the problem arises of very precisely focusing the operating laser beam onto a preselected microscopic target region typically having dimensions in the order of only one or a few microns without adversely affecting adjacent regions. This problem is difficult to solve for a number of reasons:
First, the operating laser beam is normally invisible or only intermittently visible.
Second, the operating laser beam should not be switched on before the adjustment has been made in order to avoid damage to the object by incorrect adjustment.
Third, the focusing and adjusting of the laser beam should be made without interfering with viewing the object through a microscope and its positioning in the optimum position under the microscope's objective.
Fourth, a biological object or preparation made for research of the aforementioned kind is normally completely transparent without exhibiting to the observing eye interior structures or boundaries which would serve as markings for estimating the steps of the location of a particular target region.
In the general art of using invisible or intermittently visible laser beams as a tool, for example in microsurgery, it has been common practice to make the main or working laser beam visible by superimposing on the laser beam a parallel beam of visible light, preferably a visible auxiliary laser beam. One example thereof is disclosed in U.S. Pat. No. 3,710,798 to Bredemeier where a marker beam from an He-Ne-laser is superimposed on an operating beam from a CO.sub.2 -laser and both beams are jointly focused onto a target site on the surface of a human body portion to be operated upon, said portion being viewed through a microscope. The two laser beams are focused by a focusing lens bypassing the microscope, and a further lens system is interposed in the marker beam to compensate for chromatic inaccuracies of the focusing lens and thereby to provide exact coincidence of the focal points of the two laser beams. This prior art system could not be used when microscopic areas on the order of one or a few microns in diameter are being observed and then only for a distance of a few millimeters. Also, in most cases an immersion fluid must be interposed between the object and the objective of the microscope. Moreover, the system would not help to locate the focal point of the operating laser beam at a microscopic region somewhere within a biological object where the object is completely transparent and no light from the visible marker laser beam would be scattered back through the microscope.
U.S. Pat. No. 3,096,767 to Gresser et al discloses a medical apparatus fo photo-cauterizing portions of the human body and which is adapted to produce a laser beam which can be focused by variable focusing means onto the desired part of the human body and which simultaneously can be viewed through a viewing apparatus. Four parallel visible light beams produced by an ordinary lamp and an aperture mask are superimposed on the laser beam and focused through the same variable focusing means so as to merge at a focal spot coincident with the focal spot of the laser beam. The possibility of longitudinally displacing the focal spot of the visible light beam from the focal spot of the laser beam is mentioned but means for achieving such displacement is not disclosed. This prior art apparatus too, would not be useful for adjusting the invisible laser focal spot to a very small micro-region located within a transparent object viewed under high magnification through a microscope.